Tissue Sample Preprocessing Methods and Devices

ABSTRACT

To preprocess a tissue sample, a first permeable mesh membrane is tautly stretched out over the tissue sample and overlaid on top of a flat surface. This covering sandwiches and secures the tissue sample in between the first permeable mesh membrane and the flat surface. If it is desired to reinforce the mechanical strength of the first permeable mesh membrane, a second permeable mesh membrane can be tautly stretched out and overlaid on top of the sandwich. Alternatively, the second permeable mesh membrane can be attached onto a third component, such as a disposable solvent resistant frame, to create a framed mesh. Thereafter, the first permeable mesh membrane may be attached to the framed mesh to create a compound framed mesh. The tissue sample may then be sandwiched and secured in between the compound framed mesh and the flat surface. To retain and flatten the tissue sample, gentle pressure may be uniformly applied.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of provisional patentapplication Ser. No. 61/030,452 to Eswar Prasad Ramachandran Iyer etal., filed on Feb. 21, 2008, entitled “Methods and Device for TissueProcessing,” and provisional patent application Ser. No. 61/114,289 toEswar Prasad Ramachandran Iyer et al., filed on Nov. 13, 2008, alsoentitled “Methods and Devices for Tissue Processing,” which are bothhereby incorporated by reference.

BACKGROUND OF THE INVENTION

Many commonly used research methods, including cellular imaging vialight and confocal microscopy, immunohistochemistry and fluorescent insitu hybridizations require the processing of thin and/or thickbiological tissue sections over a glass slide or a glass-coverslip. Ifthe tissue sections are well prepared and flat, and if the glass surfaceis clean, then the weak non-ionic forces, like hydrogen bonds and vander Waals forces, help the tissue sections to adhere to the glasssurface.

However, often the tissue sections are not completely flat. Also, theslide surface may contain dust particles that can prevent good surfacecontact. Moreover, some tissue types do not even adhere to a slide orglass surface at all. Because of such problems, tissue sections aregenerally lost during tissue processing, which leads to wasted time andenergy and rising costs.

For those sections that do adhere, tissue adhesion is generally notstrong enough to withstand the mechanical stress of tissue processing.Examples of mechanical stress include, but are not limited to, longincubation periods in various chemicals, high temperatures and repeatedwashing in various solutions. Although these examples may be imperativeto achieve successful processing of tissues, long processing times andrepeated physical manipulation can dislodge the tissue.

The most commonly used method for preventing the dislodging of tissuesections is by coating the glass surface with various adhesives.Examples of adhesives include poly-L-Lysine (pLL), silane, glue (e.g.,Elmer's glue), Mayere's egg albumin, chrome alum and chrome gelatin,silicon rubber, and starch paste. Another method involves creating acharged slide by using a known adhesive, such as pLL and silane.

There are a number of commercial products (e.g., adhesives and/orcharged slides) that are available in the market that are aimed atimproving the tissue retention. But, none of these completely solves theproblem. Nonlimiting examples include Tissue-Tack Adhesive (Proscitechof Queensland, Australia), Biobond Tissue Section Adhesive (SPI Suppliesof West Chester, Pa.), Superfrost Plus Gold Slides (Electron MicroscopySciences of Hatfield, Pa.), Superfrost Plus Gold Slides (Erie ScientificCompany of Portsmouth, N.H.), BD BioCoat Precoated Glass Coverslips (BDBiosciences of San Jose, Calif.), SUPERFROST PLUS—Adhesion (ElectronMicroscopy Sciences of Hatfield, Pa.), Poly-L-Lysine Coated and SilaneTreated Microscope Slide (Electron Microscopy Sciences of Hatfield,Pa.), Excell Adhesion Slides (Electron Microscopy Sciences of Hatfield,Pa.), and Polysine Microscope Adhesion Slide (Electron MicroscopySciences of Hatfield, Pa.).

It is even more challenging to mount thick tissue specimens. Forexample, a Drosophila cuticle has an uneven surface morphology and ismore prone to curling up during dehydration. For some protocols, likeimmunohistochemistry, it is essential to dehydrate the cuticle wellbefore mounting them for microscopy.

Conventionally, dehydration of the Drosophila larval cuticle isperformed by mounting the filleted, fixed, and stained larval cuticleover a poly-L-Lysine (pLL) coated coverslip. Then, it is immersedsequentially into 30%, 50%, 90%, 100% and 100% ethanol solutions (˜5minutes each). Finally, the cuticle cleared twice in xylene to removethe ethanol and make the sections optically transparent (˜10 minuteseach).

Due to the thickness and uneven surface of the larval cuticle, it isvery difficult to retain the tissues over the coverslip whilemaintaining section flatness during the dehydration process. Aconsiderable percent of tissues usually dislodge from the glass surfacedue to insufficient adhesion to the coverslip. Once dislodged, it isdifficult to recover the tissue for further analysis. Some tissues, eventhough not completely dislodged, curl in the edges, rendering them unfitfor analysis. Further, the conventional method of dehydrating thecuticle requires considerable practice and lots of handling with care tominimize the tissue loss during processing. For example, someone withjust a few months of experience in the protocol may lose about 50%-70%of total tissues processed due to poor tissue retention. Even a personwith considerable experience may lose about 20%-30% tissues regularly.The percent of tissue retained during dehydration tends also to bestrongly affected by the quality of pLL coating on the coverslips, whichmay suffer from batch-to-batch variations.

Furthermore, some methods require harsh treatment of sections (e.g.,antigen retrieval), resulting in loss of tissue. Simply, these methods,along with the ones above, cannot guarantee complete tissue retention.

Consequently, what is needed is a device that allows all kinds oftissues to be retained over a flat surface (e.g., slide, coverslip,etc.) during the tissue processing for analysis. The device should alsoprevent the tissues from loosing its flat morphology. Furthermore, thedevice should be able to withstand the harsh conditions of tissueprocessing without affecting the quality of tissue processing.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The accompanying drawings, which are incorporated in and form a part ofthe specification, illustrate an embodiment of the present inventionand, together with the description, serve to explain the principles ofthe invention.

FIG. 1 shows an example of creating a tissue samplepreprocessing/processing device.

FIG. 2 shows another example of creating a tissue samplepreprocessing/processing device.

FIG. 3 shows another example of creating a tissue samplepreprocessing/processing device.

FIG. 4 shows another example of creating a tissue samplepreprocessing/processing device.

FIG. 5 shows a chart indicating the surprising results of tissueretention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to devices, as well as methods for makingsuch devices, for preprocessing tissue samples of all kinds (i.e.,animal, human, plant, insects, etc.). These tissue sample preprocessingdevices enable one to maximize tissue sample analyses after the tissuesample has been processed.

Preprocessing refers to the mounting of one or more tissue samples on aflat surface (e.g., a glass slide, plastic slide, or coverslip) coupledwith the retention of flat, even tissue morphology on the flat surfaceas a critical parameter in preparing the tissue sample(s) forprocessing, and ultimately, microscopic visualization and analyses.

Processing refers those chemical treatments necessary to properlyprepare tissue samples for analyses. In this case for example,processing may be referred to as (1) dehydration of tissue samplesthrough a graded series of ethanol treatments coupled with xylenetreatments to optically clear otherwise opaque tissues; (2)de-paraffinization of paraffin-embedded tissue sections; and (3)performing immunohistochemistry on a flat surface that is mounting thickand/or thin tissue sections.

Preprocessing Examples

Referring to FIG. 1 as one embodiment, a tissue sample 110 to beprocessed is placed on a flat surface 105. Once placed on the flatsurface 105, a first permeable mesh membrane 115 is tautly stretched outover the tissue sample and overlaid on top of the flat surface. Tosecure the first permeable mesh membrane 115 on the flat surface 105,the edges of the first permeable mesh membrane 115 may be coated with athin lining of solvent resistant adhesive 120 (e.g., adhesive film(peelable), acrylic adhesive, solvent resistant glue, etc.) to hold thefirst permeable mesh membrane 115 onto the flat surface 105. The effectis to securely sandwich the tissue sample between the first permeablemesh membrane and the flat surface, and thus creating a first permeablemesh membrane-tissue sample-flat surface sandwich 125.

The preprocessed tissue sample in this sandwich form is now ready to beprocessed using traditional, known methods (such as air drying; passingthe samples through a chemical solution (e.g., methanol, ethanol,xylene, etc.) and/or a low surface tension solvent (e.g., Freon 113,hexamethyldisilazane); vacuuming; etc.). Additionally, tissuepreprocessed in this manner is suitable for de-paraffinization ofparaffin-embedded tissue sections and/or immunohistochemistry.

Because of the thickness of tissues tend to vary, the dimensions of thetissue sample preprocessing devices also tend to vary. As oneembodiment, the resulting dimensions of the present invention may be ˜75mm long by ˜22 mm wide by ˜1 mm thick. As another embodiment, thedimensions may be ˜75 mm long by ˜22 mm wide by ˜2 mm thick.

Flat surface 105 is defined to be any material that can be used forexamining tissue samples. Examples include a slide (glass or clearplastic), coverslip, etc. Preferably, the flat surface 105 should becleaned to remove dust, chemicals, oil, and any other foreign substancethat may obstruct tissue processing.

The first permeable mesh membrane 115 is capable of being peeled awayand discarded once tissue processing and/or analysis have beencompleted. To facilitate peeling, the first permeable mesh membrane 115may include a flap on an edge.

To aid the flattening of the tissue sample 110, pressure may beuniformly and evenly applied on the first permeable mesh membrane-tissuesample-flat surface sandwich 125. The type of pressure includespressing, compressing, thumbing, binder clipping, etc. The amount ofpressure should be a uniform gentle force. Generally, this pressure mayaid surface-to-surface contact between the tissue sample 110 and theflat surface 105. Furthermore, pressure may enhance tissue adhesionconsiderably.

The first permeable mesh membrane 115 also acts as a screen. At leasttwo resulting effects emerge from screening the tissue sample. One, thescreening acts as a protective barrier by rendering the tissue 110resistant to repeated mechanical handling and washing. Two, thescreening aids the removal of some uneven irregularities from anysection of the tissue 110 without itself inducing any irregularities orscoring of the tissue sample surface.

Overall, this tissue screening feature surprisingly allows theprocessing of tissue sections in various solutions while minimizing theeffects of stress handling on the tissues. Thus, greater tissueretention can be achieved.

Another embodiment is presented in FIG. 2. The first permeable meshmembrane 115 may be attached (via, for example, solvent resistant glueor adhesive, heat-melting, etc.) to a frame 205, such as a disposableplastic or glass frame. To allow multiple samples to be sandwiched onthe flat surface 105 and be separated from each other simultaneously,the frame 205 may have two or more compartments. Each of thesecompartments are designed to confine a sample tissue for processingand/or analysis. For instance, the frame 205 may have a split-window fortwo coverslips (e.g., ˜25 mm×˜25 mm). Alternatively, the frame 210 mayhave a single large window. The former 205 may be used for smallertissue samples 110; the latter 210 may be used for larger tissue samples110. Solvent resistant adhesive 120 may be placed along the edges tosecure the frame 205, 210 onto the flat surface 105. To further help setthe frame in place and flatten the tissue sample(s), uniform pressure215 may be applied on the frame.

Different kinds of mesh membranes with varying properties may be usedaccording to the type of tissue selected for processing (i.e.,dehydration, immunohistochemistry analysis, etc.). For instance, a meshmembrane may be hydrophilic or hydrophobic, or be neutral towards wateraffinity. Adsorption can be high, medium, low, or inert. The pHresistance can vary from ˜1 to ˜14. Some mesh may allow for high thermalstability (such as up to ˜250° C.) to withstand some tissue processingprotocols (such as antigen retrieval). Furthermore, each mesh can besterilized in different ways as well, such as irradiation or beautoclavable.

Another important feature is that the mesh membranes should be highlyresistant to chemical degradation. Furthermore, they should be heatresistant. Moreover, the mesh membranes should be strong and durable.

The mesh membranes may be made of varying types of material. Generally,they should be a fine, solvent-resistant, permeable membrane with a poresize sufficiently large to allow the free exchange of chemical andimmunohistochemical reagents. Nonlimiting examples of meshes includenylon, stainless steel, polyester, polypropylene, fluorocarbon,polyetheretherketone, Fibralon, PTFE, polyethylene, etc. Each of theabove described meshes is commercially obtainable from, for instance,Small Parts Inc. (Miramar, Fla.), which is a company specializing inmaterials for research and development.

Importantly, these meshes should also allow for the free mixing ofsolutions and reagents. To allow free mixing to occur, the pore size(i.e., pore opening diameter) of the meshes may vary from ˜10 to ˜100microns. The percentage of open area of the mesh membranes may vary from˜1% to ˜70%. In addition, the thread diameter of the mesh membranes mayvary from ˜10 to ˜200 microns.

Referring to FIG. 3, a second permeable mesh membrane 305 is introduced.In this example, the second permeable mesh membrane 305 acts mainly as amechanical support for the first permeable mesh membrane 115. Buildingonto the above exemplified first permeable mesh membrane-tissuesample-flat surface sandwich 125, the second permeable mesh membrane 305can be stretched and overlaid onto the first permeable meshmembrane-tissue sample-flat surface sandwich 125. In such case, thesecond permeable mesh membrane 305 may reinforce the durability of thefirst permeable mesh membrane 115 and simultaneously maintain theflatness of the tissue sample 110. To hold the second permeable meshmembrane 305 in place, solvent resistant adhesive 120 may be applied tothe edges prior to mounting the second permeable mesh membrane 305 ontop of the first permeable mesh membrane 115. Uniform gentle pressure215 may then be applied to contact the second permeable mesh membrane305 with the first permeable mesh membrane-tissue sample-flat surfacesandwich 125. The result may be called a second permeable meshmembrane-first permeable mesh membrane-tissue sample-flat surfacesandwich 310.

The second permeable mesh membrane 305, as well as any additional meshmembranes that may be used, can be of the same or equivalent types asthe first permeable mesh membrane. Hence, as one embodiment where thedevice implements multiple mesh membranes, the first permeable meshmembrane is a fine Nitrex nylon mesh and the second permeable meshmembrane is a coarse nylon mesh. The sieve size of the fine Nitrex nylonmesh may vary from ˜10 μm to ˜100 μm. As one aspect, the first permeablemesh membrane has a sieve size of ˜30 μm.

With respect to the coarse nylon mesh, it should have a relatively largesieve size. For instance, the selected sieve size may be ˜300 μm.However, the sieve size may range anywhere from ˜100 μm to ˜500 μm. Theopen area may be ˜50%. For a coarse nylon mesh having a size of ˜30cm×˜30 cm, the thickness may be ˜200 μm.

In yet another embodiment, as shown in FIG. 4, a third component 405 maybe integrated. Examples of the third component include a durable,solvent resistant metallic frame, durable solvent resistant plasticframe, a disposable slide unit with or without closeable flaps, etc.Where the third component 405 is a solvent resistant metallic frame, thedimensions of the metallic frame may be, for example, ˜75 mm long×˜25 mmwide×˜1 mm thick, with an inner opening of ˜40 mm long×˜15 mm wide.

The second permeable mesh membrane 305 may be attached over thirdcomponent 405 to create a framed mesh 410. To adhere the secondpermeable mesh membrane 305 to such metallic frame 405, a solventresistant adhesive 120 (e.g., acrylic, cyanoacrylamide, solventresistant glue, etc.) may be used. To further secure the adhered secondpermeable mesh membrane 305 onto the third component 405, the adheredsecond permeable mesh membrane 305 may also be heat-melted onto thethird component 405. Thereafter, the first permeable mesh membrane 115may be stretched, glued, and heat-melted over the second permeable meshmembrane 305 to create a compound framed mesh 415. This compound framedmesh 415 may then be used to sandwich the tissue sample onto a flatsurface, creating a first permeable mesh membrane-second permeable meshmembrane-third component-tissue sample-flat surface sandwich 420.

Processing Examples

After mounting the tissue sample using any of these embodiments(preprocessing steps), the tissue sample may be processed by placing theassembly in a container of required dimensions (for example, a 50 mlconical tube) containing the appropriate solution and left undisturbedor over a gyrating nutator mixer or a shaker for a prescribed incubationtime.

Then, after each incubation, if required, the excess solution may bedrained from the assembly by tilting and touching one corner over anabsorbent tissue. Independent of tissue type, once the protocol iscomplete, the membrane(s) and/or frame may be gently pulled apart,leaving the tissues intact over the flat surface. The flat surface withthe tissues may then be removed and be subjected to further analysis(e.g., microscopic imaging, etc.).

For instance, for dehydration of Drosophila larval cuticle, anapproximate incubation time of ˜5 to ˜10 minutes in each ethanolconcentration and xylene solution is required. Incubation times may varywidely depending on the tissue type, reagents used, and the specificprotocol implemented.

A second example involves the processing of human tissue samples, suchas those from normal or diseased tissue that has been embedded fortissue sectioning (thick or thin) in paraffin. Human tissue samplesinclude, but are not limited to, breast, lung, heart, colon, prostate,epidermis, blood vessels, stomach, intestines, kidney, eyes, brain, etc.Once paraffin-embedded tissue sections are mounted on a flat surface,these tissue sections can be processed using any of the above methodsand devices. A nonlimiting example of processing includes an initialde-paraffinization of tissue section necessary for subsequent tissueprocessing procedures. This stage of tissue processing tends to behighly susceptible to tissue dislodgement from the mounting surface.Upon successful de-paraffinization, the tissue sections mounted on thedevice can then be processed for standard immunohistochemistry analysesand, where appropriate, for ultimate microscope visualization.

Advantages

These methods of tissue retention offer numerous advantages over theconventional methods of tissue retention. First, as in the case ofretaining tissue from Drosophila, as shown in FIG. 5, the improvedmethods generally offer nearly 100% tissue retention (such as during thedehydration step of a Drosophila larval cuticle for performingimmunohistochemical analysis). These surprising results sharplycontrasts results generally obtained through conventional methods ofmounting Drosophila larval cuticle during immunohistochemistry, whichoften offer only ˜70%-˜75% tissue retention, even by an experienceduser, and suffer from batch to batch variations in pLL coating of theslide or coverslip.

Second, little to no previous experience is required for mounting andprocessing tissue samples successfully. Even an inexperienced user canaccomplish nearly 100% tissue retention during tissue processing byusing this new method. But, under the conventional way, considerableamount of training and experience is required. Users with just a fewmonths of experience may only be able to retain ˜30% to ˜40% of thetissues using the conventional method.

Third, the new way improves tissue quality by allowing mild agitationduring processing to enhance fluid mixing, such as during thedehydration process of Drosophila larval cuticle prior to imaginganalysis or in tissue de-parafinization and immunohistochemistry ofhuman tissue samples. The conventional method creates poor mixing offluids during dehydration.

Fourth, the new method is highly tolerant to mechanical handling duringtissue processing. The conventional method offers very mild tolerance tomechanical handling due to poor adhesion to the slide or the coverslip.

Fifth, efficiency in the new method remains unaffected by batch-to-batchvariation of pLL coating quality. On the contrary, efficiency in theconventional method is usually affected by batch-to-batch variation ofpLL coating quality.

Sixth, the new method enables very flat tissue sections, with minimaldeformation during dehydration. The tissue sections using theconventional method, however, are prone to mechanical deformation, suchas curling and lipping edges.

In essence, the above described tissue processing device prevents tissueloss during the dehydration of tissue samples, such as a Drosophilalarval cuticle, as well as in the processing of thick or thinparaffin-embedded tissue sections for immunohistochemistry. Such lossprevention save considerable amount of time, money, and preciousreagents by increasing the efficiency of processing. The embodieddevices are very user-friendly and do not require extensive training orany previous experience. The membrane composition, pore size, and thegeometry of the device can be modified and optimized for otherapplications, which demand tissue retention over a flat surface forvarious types of tissue processing.

The foregoing descriptions of the embodiments of the claimed inventionhave been presented for purposes of illustration and description. Theyare not intended to be exhaustive or be limiting to the precise formsdisclosed, and obviously many modifications and variations are possiblein light of the above teaching. The illustrated embodiments were chosenand described in order to best explain the principles of the claimedinvention and its practical application to thereby enable others skilledin the art to best utilize it in various embodiments and with variousmodifications as are suited to the particular use contemplated withoutdeparting from the spirit and scope of the claimed invention. In fact,after reading the above description, it will be apparent to one skilledin the relevant art(s) how to implement the claimed invention inalternative embodiments. Thus, the claimed invention should not belimited by any of the above described example embodiments. For example,the claimed invention may be practiced over other types of tissue andunder different clinical conditions (such as processing of human oranimal tissue samples).

In addition, it should be understood that any figures, graphs, tables,examples, etc., which highlight the functionality and advantages of theclaimed invention, are presented for example purposes only. Thearchitecture of the disclosed is sufficiently flexible and configurable,such that it may be utilized in ways other than that shown. For example,the steps listed in any flowchart may be reordered or only optionallyused in some embodiments.

Further, the purpose of the Abstract is to enable the U.S. Patent andTrademark Office and the public generally, and especially thescientists, engineers and practitioners in the art who are not familiarwith patent or legal terms or phraseology, to determine quickly from acursory inspection the nature and essence of the claimed invention ofthe application. The Abstract is not intended to be limiting as to thescope of the claimed invention in any way.

Furthermore, it is the applicants' intent that only claims that includethe express language “means for” or “step for” be interpreted under 35U.S.C. §112, paragraph 6. Claims that do not expressly include thephrase “means for” or “step for” are not to be interpreted under 35U.S.C. §112, paragraph 6.

A portion of the claimed invention of this patent document containsmaterial which is subject to copyright protection. The copyright ownerhas no objection to the facsimile reproduction by anyone of the patentdocument or the patent invention, as it appears in the Patent andTrademark Office patent file or records, but otherwise reserves allcopyright rights whatsoever.

1. A tissue sample preprocessing device comprising: a. a first permeablemesh membrane; and b. at least one flat surface; and wherein the firstpermeable mesh membrane i. is tautly stretched out over a tissue sampleand overlaid on top of the flat surface, sandwiching and securing thetissue sample in between the first permeable mesh membrane and the flatsurface; and ii. has a sieve size of approximately 10 μm toapproximately 100 μm.
 2. The device according to claim 1, whereinpressure is applied to the first permeable mesh membrane to flatten thetissue sample.
 3. The device according to claim 1, wherein the firstpermeable mesh membrane is a fine Nitrex nylon mesh membrane.
 4. Thedevice according to claim 1, further including a second permeable meshmembrane being tautly stretched out and over the sandwiched tissuesample.
 5. The device according to claim 4, wherein the second permeablemesh membrane is a coarse nylon mesh membrane.
 6. The device accordingto claim 4, wherein the second permeable mesh membrane has a sieve sizeof approximately 300 μm.
 7. The device according to claim 1, furtherincluding a framed mesh comprising a second permeable mesh membraneattached to a third component, the third component being a durable,solvent resistant frame.
 8. The device according to claim 7, wherein thefirst permeable mesh membrane is attached to the framed mesh to create acompound framed mesh.
 9. The device according to claim 8, wherein thetissue sample is tautly sandwiched and secured in between the firstpermeable mesh membrane of the compound framed mesh, and the flatsurface.
 10. A method for preparing tissue samples for processingcomprising sandwiching and securing a tissue sample in between a firstpermeable mesh membrane having a sieve size of approximately 10 toapproximately 100 μm and a flat surface, wherein the first permeablemesh membrane is tautly stretched over the tissue sample and over theflat surface.
 11. The method according to claim 10, wherein the firstpermeable mesh membrane is a fine Nitrex nylon mesh.
 12. The methodaccording to claim 10, further including wrapping a second permeablemesh membrane around the sandwiched tissue sample.
 13. The methodaccording to claim 12, wherein the second permeable mesh membrane is acoarse nylon mesh with a sieve size of approximately 300 μm.
 14. Themethod according to claim 10, further including attaching a secondpermeable mesh membrane to a third component to create a framed mesh,the third component being a durable, solvent resistant frame.
 15. Themethod according to claim 14, wherein the first permeable mesh membraneis attached over the framed mesh, creating a compound framed mesh. 16.The method according to claim 15, wherein the tissue sample is tautlysandwiched and secured in between the first permeable mesh membrane ofthe compound framed mesh, and the flat surface.